A method of perfusing the isolated human colon in vitro was developed to study the effect of the short-chain fatty acid n-butyrate on sodium absorption under controlled conditions. The isolated colon was viable in vitro provided that ischemia to the colon prior to perfusion was less than 40 min. Viability was judged on glucose utilization, mucosal potential difference, an sodium absorption. Sodium absorption from the lumen was observed either with or without 20 mM n-butyrate. In a control group sodium absorption (nmol/min/cm2 /+- SEM) was 320 /+- 10 (four perfusions, nine observation intervals) and potassium secretion 26 /+- 3 (four perfusions, nine observation intervals). With 20 mM n-butyrate sodium absorption was 1960 /+- 480 (four perfusions, ten observation intervals) (P less than 0.0025). Potassium secretion was 72 /+- 2 (four perfusions, ten observation intervals) and (P less than 0.025). Butyrate absorption was 254 /+- 60 (four perfusions, ten observation intervals) and correlated linearly with the unidirectional flux (Jm leads to S) of sodium (linear coefficient of 0.714, P = less than 0.001). These results suggest that the presence of bacterial short-chain fatty acids may determine the efficiency of sodium absorption in the colon and also indicate that an absence of short-chain fatty acids in the colon could be one factor leading to diminished sodium absorption in the colon of man.